Copper is an essential trace element with a critical role in the biochemistry of cellular respiration, antioxidant defense and iron homeostasis. The liver is the central organ of copper homeostasis in human and the long-term objective of these studies is to define the role of copper in pediatric liver disease. Recent studies have revealed that the delivery of copper to specific proteins within hepatocytes is mediated by distinct intracellular carrier proteins termed chaperones. The studies in this proposal are intended to elucidate the role of the copper chaperone HAH1 in copper trafficking and metabolism in hepatocytes. Structure/function studies will be accomplished utilizing site-directed mutagenesis and expression of HAH1 in Saccharomyces cerevisiae. The role of HAH1 in copper delivery to the secretory pathway will be examined by protein-protein interaction studies with the Wilson disease ATPase and the interaction of these proteins in intact single cell swill be examined using green fluorescent fusion proteins and fluorescence resonance energy transfer. The precise function of HAH1 in mammalian cells will be determined by analysis of copper trafficking and metabolism in the hepatocytes of mice with a targeted germline deletion of the murine HAH1 gene. Finally, the role of HAH1 in pediatric liver disease will be studied by HAH1 sequence analysis in genomic DNA from children with liver disease characterized by copper accumulation and toxicity including individuals diagnosed with Wilson disease but without detectable mutations in the Wilson ATPase. Taken together the results of these studies will permit further insight into the molecular and cellular mechanisms of copper trafficking in hepatocytes and may allow for novel therapeutic approaches to prevent or ameliorate childhood liver disease resulting from perturbations in metal metabolism.